In this Project, our goals are to investigate the pathogenic mechanisms of Chlamydia trachomatis by examining how these obligate intracellular pathogen regulates it unusual dimorphic cycle, and by identifying novel virulence genes and mechanisms. The objective of this application is to apply genetic techniques to the investigation of developmental gene regulation and chlamydial pathogenesis. One hypothesis to be tested is that gene regulation or repression at critical phases of the life cycle reflects a response to environmental signals which trigger differentiation between the vegetative reticulate body (RB) and the metabolically inert but infectious elementary body (EB) prior to release from the eukaryotic cell. In addition, we aim to generate allele-replacement mutants of C. trachomatis which can be studied in suitable animal models of chlamydial infection and disease. The rationale behind this research is that the development of techniques for the genetic manipulation of C. trachomatis will lead to methods of examining virulence gene expression and regulation in vivo. To accomplish the objectives of this application we will pursue two specific aims: (i) to identify and characterize temporally regulated promoters in vivo, focusing particularly on those that are of strongly up regulated late in the developmental cycle, (ii) to develop a generally applicable method for the direct selection of gene replacement mutants in Chlamydia. At the completion of this research we expect to have identified genes which are involved in differentiation of RBs to EBS. We also expect to have derived a system for site-specific mutagenesis of C. trachomatis and to have demonstrated its efficacy by generating site-specific mutations in genes which have been suggested to play a role in the pathogenesis of chlamydial disease.